Preformed polymer coating process and product

ABSTRACT

A method of coating a solid support (e.g. a capillary or chromatography packing) to alter the properties of the support surface for separating components in a fluid stream. The method comprises (a) covalently binding a coupling agent (including functional groups capable of forming free radical sites under hydrogen abstraction conditions) to the support surface in a uniform layer, and (b) thereafter, contacting the bound coupling agent with a solution of preformed polymer comprising totally saturated carbon chain backbones including leaving groups, under hydrogen abstraction conditions of elevated temperature in the presence of a free radical catalyst to remove leaving groups from the carbon chains to form free radical carbon binding sites which covalently bond to the coupling agent layer and to crosslink at least some of the preformed polymer through the free radical carbon binding sites to form a dimensional polymer network coating on said solid support surface. Alternatively, the coating is applied directly to an organic solid support without an intermediate coupling agent.

BACKGROUND OF THE INVENTION

Capillary Electrophoresis (CE) has emerged as an important tool foranalyzing biomolecules. The high efficiency, high resolution, andautomation capabilities of CE make it highly suitable in the routineanalysis of proteins, peptides, and even small ions. A major problemencountered in the above separations is the interaction of basicanalytes, such as basic proteins, with exposed surface silanol groups onthe capillary wall. This interaction results in a loss of efficiency andirreproducible separations. Typical approaches in addressing the aboveproblem include working at conditions where the silanol groups areeither un-ionized¹ or fully ionized². These conditions, however, entailworking at extremes of pH and may be unsuitable for many analytes.Additionally, silica dissolves at extreme pH's, which is anotherlimitation of this approach.³ Other approaches in addressing the aboveproblem involve adding compounds⁴⁻⁶ that compete with the analytes forinteraction sites on the capillary wall. These additives, however, mayadversely affect the separation of analytes.

Another popular approach includes working with coatings that are eitherphysically adsorbed or chemically attached to the capillary surface.⁷⁻²¹These coatings mask the presence of surface silanols and enhanceseparation efficiency. The adsorbed coatings suffer from limitedstability and require repeated replenishment for effective operation⁷.Recently, Gilges et al.⁷ showed excellent separation of basic proteinsusing a polyvinylalcohol (PVA) coated capillary. The polymer coating wasachieved by a thermal treatment that immobilized PVA on the capillarywall. This coated capillary gave a low electroosmotic (EO) flow up to pH9. However, only 40 runs were possible at pH 8.5 without loss ofefficiency. Buffers such as borate, Tris HCl, and Tri-phosphate did notprovide good separation of proteins using this coated capillary, thuslimiting its utility.

A review of coatings for CE reveals several examples of chemicallymodified capillaries that were designed to minimize the presence ofsurface silanols and reduce analyte interactions. These modificationsinvolve attaching or creating one or more polymeric layers on thesurface of the capillary through various coupling chemistries. In 1985,Hjerten⁸ showed a two-step coating process by attaching a bifunctionalsilane on the surface of the capillary followed by in situpolymerization of a vinyl group containing monomer. The presence of apolymerizable C═C group was essential in both the monomer and silane forcoupling. Strege and Lagu⁹ showed that the above coating gives a verylow EO flow, but achieved poor separations of a mixture of proteins. Thepoor peak shapes obtained with this capillary were attributed toelectrostatic and/or hydrogen bonding interactions of the proteins withthe capillary wall or coating. It was necessary to incorporate asurfactant in the CE run buffer to achieve good separations of proteins.Similarly, a cross-linked in situ polymerized polyacrylamide capillarygave poor separation efficiencies for basic proteins when tested with noadded cationic additives in the buffer¹⁰.

As an alternative approach to in situ polymerization, coatings areformed by reacting silanes that have appropriate reactive end groupswith reactive end groups on prederivatized polymers. These coatings weredisclosed by Herren et al.¹¹ to mininize or reduce EO flow. Theydiscussed several synthetic procedures for creating various derivativesof dextran¹² and PEG¹³ and their utility in several applicationsincluding modifying control pore glass beads. However, data on the pHstability of this coating and its performance with proteins as testanalytes were not shown. Following a similar approach, Hjerten andKubo¹⁴ showed the attachment of several polymers (e.g., methylcelluloseand dextran) after a prederivatization step. The prederivatization stepwas required prior to attaching the polymers to the methacryl silanetreated capillary. Additionally, the polymer coupling process wasdependent upon a high yield of the prederivatization reaction.

Recently, Malik et al.¹⁵,16 adapted a GC-type static coating procedure,in which the coating was achieved by depositing a mixture of polymer,initiators, and silane reagent on the surface of a capillary by using alow boiling point solvent. The capillary was then heat treated tocross-link the surface film. The coating thickness influenced the EOflow and performance and required optimization. In comparing data fromMalik et al.,¹⁵,16 variabilities in efficiencies were observed betweenanalytes in a Superox-4 coated capillary and between two Superox-4coated capillaries. Similarly, two Ucon 75-H-90000 polymer coatedcapillaries tested under identical conditions gave different migrationtimes and mobilities, indicating problems with the reproducibility ofthe coating process.

The above coatings were attached through Si--O--Si--C linkage. Toovercome the limited pH stability of the Si--O--Si bond, severalresearchers used approaches such as attaching polyacrylamide by in situpolymerization through a Si--C linkage¹⁷ and attaching a hydrolyticallystable derivative of acrylamide by in situ polymerization.¹⁸ Theseapproaches enhanced the coating stability relative to Hjerten's originalapproach and provided better efficiencies for basic proteins. However,multiple reaction steps with stringent conditions were required duringthe coating process. For example, the approach by Cobb et al.¹⁷ requiredanhydrous solvents and conditions during the Grignard reaction step.Similarly, the work by Chiari et al.¹⁸ required synthesis of a specialmonomer to achieve a stable and efficient coating. Other approachesinvolved cross-linking or attaching several polymeric layers on thecapillary surface. Increased coverage on the capillary surface by thevarious polymeric layers was expected to diminish any interaction of theanalytes with the exposed surface silanols. Smith et al.¹⁹ showedseparations of proteins in coated capillaries that had a primary silanelayer anchored to several polymeric layers. Some layers were adsorbed ontop of the primary layer. Huang et al.²⁰ showed separation of proteinsusing a cross-linked, immobilized, hydrophilic polymer layer atop ahydrophobic, self assembled, alkyl silane layer. Schmalzing et al.²¹showed excellent separations of basic proteins in a multilayeredcross-linked coated capillary. In situ polymerization of a monomer ontop of a cross-linked primary silane layer resulted in a hydrophilicpolymeric layer that was subsequently cross-linked. The above approacheswere all multistep processes and, in some cases, required additionalcross-linking steps.²¹

There is a need for a simple method for coupling preformed underivatizedpolymers covalently to the surface of a conduit such as a fused silicacapillary used for capillary electrophoresis.

In addition, polymer based support surfaces have been used forseparating the components in a fluid stream such as for capillaryelectrophoresis or liquid chromatography. Such polymeric supportsurfaces can be on the inner walls of the conduit (e.g. capillary), orcan form a packing of polymeric particles for liquid chromatography. Insome instances, such polymeric support surfaces do not have the desiredproperties for separating components. For this purpose, such surfaceshave been modified by coating with suitable hydrophilic polymers asdisclosed in Afeyan et al. (U.S. Pat. No. 5,503,933).

SUMMARY OF THE INVENTION

An object of the invention is to provide a cross-linked coating ofpreformed polymer directly or indirectly covalently linked to thesurface of the support. In one embodiment, an intermediate couplingagent is used between the support surface and the preformed polymerwhile, in the other embodiment, it is not.

Referring first to the coupling agent embodiment, a solid supportaccording to the present invention has a coating on its surface whichalters the properties of the support surface for separating componentsin a fluid stream in contact therewith. The coating comprises a couplingagent including a functional group and is covalently bound to saidsupport surface in a substantially uniform layer, and a preformedpolymer comprising totally saturated, substituted or unsubstituted,carbon chain backbones from which leaving groups have been abstractedwhile in solution and in contact with said coupling agent layer to formbonding sites on said preformed polymer which covalently bind to saidcoupling agent and which crosslink said preformed polymer forming acoating comprising a three-dimensional, cross-linked polymer network onsaid solid support.

The invention also includes a method of coating the solid supportsurface which alters the properties of the support surface forseparating components in a fluid stream. The method comprises (a)covalently binding a coupling agent (including functional groups capableof forming free radical sites under hydrogen abstraction conditions) tosaid support surface in a uniform layer, and (b) thereafter, contactingsaid covalently bound coupling agent with a solution of said preformedpolymer comprising totally saturated, substituted or unsubstituted,carbon chain backbones including leaving groups, under hydrogenabstraction conditions of elevated temperature in the presence of a freeradical catalyst, said support surface not being soluble in saidpolymer, to remove leaving groups from said preformed polymer carbonchains to form free radical carbon binding sites which covalently bondto said coupling agent layer and to crosslink at least some of saidpreformed polymer through thus-formed free radical carbon binding sitestherein to form a dimensional polymer network coating on said solidsupport surface.

In one instance, the second functional group in the coupling agent istypically a carbon moiety bound to a leaving group such as a reactivegroup (e.g. a halogen) or hydrogen. The leaving group is capable ofbeing abstracted to a free radical under hydrogen abstractionconditions. For a coupling agent including carbon chain, the reactivegroups or hydrogen may be terminal groups or interior of the carbonchain. In another embodiment a coupling agent including a carbon chainincludes a second functional group in the form of unsaturation in thecarbon chain (e.g. a terminal double bond) (C═C). Such unsaturatedgroups react with the free radical sites on the preformed polymer by afree radical addition reaction.

In the above method and coated solid support, one preferred supportsurface comprises silica with a coupling agent comprising a silane.Preferred leaving groups are hydrogen or halogens. The solid supportpreferably is either the inner wall of a capillary for capillaryelectrophoresis or the packing of a flow-through particle bed such asone used for liquid chromatography.

In another embodiment of the invention, no intermediate coupling agentis used for coating an organic polymeric solid support which hassaturated or unsaturated carbon chains including functional groupscapable of forming free radical sites under hydrogen abstractionconditions to alter the properties of separating components in a fluidstream in contact therewith. The coating comprises preformed polymerincluding totally saturated substituted or unsubstituted carbon chainbackbones from which leaving groups have been abstracted while insolution and in contact with said support surface to form free radicalbinding sites on said preformed polymer and covalently bound to freeradical binding sites formed on said support surface functional groups,and to crosslink said preformed polymer forming a coating comprising athree-dimensional, cross-linked polymer network on said solid support.

One preferred method for coating the polymeric solid support surfacecomprises contacting said support surface with preformed polymercomprising totally saturated carbon chain backbones including leavinggroups under hydrogen abstraction conditions of elevated temperature inthe presence of a free radical catalyst, to abstract hydrogen or otherleaving groups from said preformed polymer carbon chains to formabstracted carbon sites which covalently bond said support surface tocarbon chains, and to crosslink at least some of said preformed polymerthrough free radicals created at said hydrogen extraction carbon sitestherein to form a three-dimensional polymer network coating on saidsolid support surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 are electropherograms showing separations of basic proteins usingvarious polymer coated capillaries.

FIG. 2 is a reproducibility study using a MET-PVP (360K) coatedcapillary.

FIG. 3 is a series of graphs slowing the effect of polymer molecularweight and concentration on efficiency of basic proteins.

FIG. 4 is a electropherogram illustrating acidic protein separationsusing a cationic polymer coated capillary.

FIG. 5 is a electropherogram illustrating separation of test anionsusing a cationic polymer coated capillary.

FIG. 6 is a electropherogram illustrating separation of proteins from 2%Vitamin D Milk using a MET-PVP coated capillary.

FIG. 7 is a electropherogram illustrating separation of hemoglobinvariants using a MET-PVP coated capillary.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is directed to modifying the properties of a solid supportsurface for separating components in a fluid stream in contact with suchsurface. In one embodiment, the support surface comprises the inner wallof a conduit such as a capillary for capillary electrophoresis orcapillary electrochromatography or capillary liquid chromatography orsupercritical fluid chromatography or gas chromatography. In anotherembodiment, the solid support surface comprises the particulate packingof a flow-through particle bed used for liquid chromatography orcapillary electrochromatography or supercritical fluid chromatography orgas chromatography. Other type of solid support surfaces useful inelectrophoresis which could be modified according to the inventioninclude silica or glass based microfabricated capillary array systemsand slab-gel systems.

The invention will first be described with respect to the embodiment inwhich a coupling agent is used and in which the solid support surface isthe inner wall of a capillary for capillary electrophoresis. However, itshould be understood that the coupling agent mode could be used forother solid surfaces as described above.

A wide variety of solid support surfaces can be used so long as they arecapable of covalent binding to a coupling agent which in turn is capableof covalent binding to a preformed polymer under hydrogen abstractionconditions. A common material used for the capillary in capillaryelectrophoresis is fused silica. Like other suitable support surfaces,it contains hydroxyl groups which are readily coupled to preferredcoupling agents such as silanes. Suitable inorganic solid supportsurfaces containing hydroxyl groups, or groups convertible to hydroxylgroups, include silica, titania, quartz, glass, alumina, thoria,beryllia and zirconia.

Suitable coupling agents are ones which are capable of covalent bindingto such support surfaces and, in turn, of covalent binding to thepreformed polymer under hydrogen abstraction conditions as describedbelow. Mechanisms of attachment of coupling agents to common supportsurfaces such as silica are well known such as illustrated in HjertenU.S. Pat. No. 4,680,201. These coupling agents typically arebifunctional compounds with a first functional group capable of covalentattachment to the solid support surface and a second functional group(in the form of a leaving group) capable of binding to the preformedpolymer. Suitable coupling agent first functional groups capable ofbinding the solid support include mono, di and tri alkoxy groups such asmethoxy and ethoxy groups and halogens such as chlorine. As described,after one of the functional groups of the coupling agents is bound tothe support surface, the coupling agent still includes a secondfunctional group capable of forming a free radical binding site whichcovalently binds the preformed polymer. Such second functional groupcould be a saturated carbon chain in which the bound hydrogen is aleaving group. Alternatively, the second functional group could be aleaving group such as hydrogen or a halogen. Moreover, the secondfunctional group could be unsaturation, e.g. in the form of C═C bondscapable of forming free radicals under hydrogen abstraction conditions.For purposes of the present invention, all of such coupling agent groupscapable of binding the preformed polymer under hydrogen abstractionconditions are termed "second functional groups".

In the present process, the free radical binding sites are formed at thecoupling agent second functional groups under hydrogen abstractionconditions by the removal of a leaving group or breaking of a C═C doublebond. At the same time, the preformed polymer in solution also forms afree radical binding site in contact with the coupling agent freeradical binding site to form a covalent bonding between the couplingagent layer and the preformed polymer.

The principles that govern the lability or ease of release of leavinggroups are well known. Encyclopedia of Polymer Science and Engineering,Vol. 13, p.818, Lenz, R. W. Organic Chemistry of Synthetic HighPolymers, (1967) pp. 288-289, and Encyclopedia of Polymer Science &Engineering, Vol. 13, p.714. For example, release of steric compressionon radical formation partially accounts for the progressive decrease inthe strength of C--H bonds from primary to secondary to tertiary.Important labile hydrogen groups include protons on the carbonylfunctions of aldehydes and formate esters, on the carbinol functions ofprimary or secondary alcohols, on the α-carbon atoms of amines andethers, on the thiol functions of mercaptans and on carbon atomsadjacent to unsaturated functions. Abstraction of leaving groupabstraction follows the order of strength of the bonds being broken,except for abstraction of hydrogen:

    I>Br˜H>Cl>F

Chlorine, bromine and iodine are very labile (e.g. in polyhalomethanesand other haloalkanes).

One preferred form of coupling agent is a silane. Suitable silanes ingeneral can be designated as R.sub.(n) SiX.sub.(4-n). Here, the X groupreacts with the substrate and this results in a covalent bond betweenthe substrate and the silane. X is a hydrolyzable group and includesmono, di or tri substituted alkoxy such as methoxy or ethoxy, or halogengroups such as chlorine. The R group consists of a non-hydrolyzableorganic chain which includes end-group fluctionalities such as acetoxyor acryloxy or allyl or amino or alkyl or benzyl, or vinyl groups. R mayalso includes other suitable functionalities such as halo or cyano orthiocyano or mercapto groups. Suitable silanes are listed in 1) SiliconCompounds: Register & Review, from United Chemical Technologies, 5thEd., 1991, and in (2) Tailoring Surfaces with Silanes, Chemtech 7, 766(1977). They include allyltrimethoxysilane, chlorodimethyloctylsilane,γ-methacryloxypropyltrimethoxysilane, 3-aminopropyltrimethoxysilane and3-glycidoxypropyltrimethoxysilane.

The coating process described herein requires no reactivefunctionalities for coupling, either on the polymer or on the silane.Free radical sites formed on both the polymer and the silane aresufficient for simultaneous coupling and cross-linking. No specialconditions are required for this coupling reaction. The capillariescoated by the above process satisfy the four major requirements of acoated capillary for CE; namely: (1) provide reproducible separation ofanalytes through generation of reproducible EO flow; (2) allow minimalinteraction with the analytes, thus maximizing efficiency of theseparation and recovery, (3) show minimal absorbance at the monitoredwavelength for enhanced sensitivity, and (4) retain stable performanceunder a variety of buffer and pH conditions for robust operation.

As is well recognized, a silane coupling agent bound to a silica solidsupport surface forms Si--O--Si linkages. Silicon Compounds: Registry &Review from United Technologies, 5th Ed. p.59. However, coupling agentsother than silanes may be employed to provide different couplinglinkages. For example, an allyl methacrylate coupling agent may be boundto a silica surface by Si--C linkage¹⁷. Other coupling agents withsilica walls include an alcohol (after treatment with thionyl chloridereagent) which form a Si--O--C linkage (Snyder, L. R. & Kirkland, J. J.,Introduction to Modern Liquid Chromatography, John Wiley & Sons, Inc.,1979, Chapter 7, p.272-3). Yet, another coupling agent of the amine typeproduces Si--N linkage (after treatment with thionyl chloride reagent).Snyder, Supra. Analogous linkages are formed between silanes andhydroxyl-containing solid support surfaces other than silica (e.g.Ti--O--Si) (Matyska et al., in Poster #P-0561, 20th InternationalSymposium of High Performance Liquid Phase Separations and RelatedTechniques, June 1996, San Francisco, Calif.).

The preformed polymer of the present invention is coupled to the freeradical sites formed at least in part by removal of leaving groups inthe preformed polymer backbone under hydrogen abstraction conditions.The same principles apply to selection of suitable leaving groups forthe coupling agent as for the preformed polymer. The preformed polymeris formed of totally saturated carbon chain backbones which have notbeen prederivatized to form unsaturation prior to covalent bonding witha coupling agent. However, the preformed polymer may includeunsaturation in carbon side chains or moieties, such as aromatic groups,including benzene rings, e.g. in polystyrene. The carbon chain backbonestypically include from 50 to 10,000 carbons in the chains. They may beof any suitable length to provide the desired property so long as theyinclude leaving groups which are abstracted under hydrogen abstractionconditions to form covalent bonds with the coupling agent which haspreviously been covalently bound to the support surface.

For a smooth or regular surface such as the inner wall of a capillary,the coupling agent binds in a substantially uniform layer to the supportsurface. That is, it substantially covers the support surface byinteracting with the available reactive sites on such surface when it isuniformly available as the inner wall of a capillary. If the surface isirregular such as the pores of a macroporous surface, there would becorresponding irregularity in the uniformity of the coating.

The carbon chains, typically polymerized, of the preformed polymer maybe unsubstituted, i.e. include only hydrogen groups along the chainwhich are abstractable under hydrogen abstraction condition to form freeradical sites. Alternatively, the carbon chains may be substituted by avariety of groups or moieties which can serve different purposes. Forexample, the group may comprise leaving groups (e.g. halogen) which areabstracted to form the free radical bonding sites for bonding to thecoupling agent and for cross-linking. Other substituted leaving groupsinclude hydrogen bound to sulfur as in mercapto "SH" groups typically atthe end of the chain. Also, the substitution may comprise non-leavinggroups which serves the function of altering the properties of thesupport surface for separating the components in the fluid stream. Forexample, a quaternary nitrogen atom imparts ion exchange selectivity tothe support surface after it is bound. Similarly, an OH group impartshydrophilicity to the support surface after it is bound.

A suitable list of preformed polymers which include appropriate leavinggroups and also the ability to alter characteristics include substitutedor unsubstituted polyalkylenes, polyesters, polyamines, polyamindes,polyethers, polysufonates, polyoxides, polyalkyleneglycols, polystyrenicbased polymers, polyacetals, polychlorides, polysaccharides,polycarbonates, polymers of monoethylenically unsaturated monomers,polymers of polyvinylidene monomers and mixtures and copolymers of theabove polymers. Preferred suitable specific preformed polymers includepolyethyleneoxide, polyacrylamide, polyvinylalcohol,polyvinylpyrolidone, polyethyleneglycol, acrylamidomethylpropylsulfonicacid, polyacrylic acid and methacrylamidopropyltrimethylammoniumchloride.

Other characteristics of the preformed organic polymer are that they besufficiently soluble in solvent to uniformly coat the solid supportsurface without dissolving the solid support. Suitable solvents includewater, alcohols such as methanol, ethanol, polyols such as glycerine,ethylene glycol, ketone-alcohols such as diacetone alcohol, acids suchas formic acid, acetic acid, ether-alcohols such as glycol ethers,lactones such as γ-butryolactone, esters such as ethyl lactate, ethylacetate, ketones such as methylcyclohexanone, acetone, chlorinatedhydrocarbons such as methylene dichloride, chloroform, carbontetrachloride, lactams such as 2-pyrolidone, N-methyl-2-pyrolidone,amines such as butylamine, cyclohexylamine, aniline, morpholine,nitroparaffins such as nitromethanes, hydrocarbons such as benzene,toluene, hexane, alone or in combination with other solvents, etherssuch as dioxane, tetrahydrofuran, chlorofluoroalkanes such asdichloromonofluoromethane, inorganic solutions of salts such asaluminium potassium sulfate, ammonium chloride, ferric chloride, sodiumchloride, potassium chloride, etc.

The separation characteristic of the coating can be used in a widevariety of applications. For example, it could be used in capillaryelectrophoresis in which the coating can vary EO flow. The coating canbe anionic, cationic or neutral, depending on the desired effect. Thecoating substantially covers the solid surface. It covalently bonds in across-linked three-dimensional matrix between preformed polymer chainsand with the coupling agent. This cross-linked coating is highly stable.It is stable over a wide pH range (e.g. 2 to 10) due to its high levelof cross-linking on the surface. The extent of substitution on thepolymer chain and on the coupling agent (e.g. silane) together with typeof solvent and initiator system,²² determines the extent ofcross-linking and coupling.

Referring back to the method of formation, the coupling agent is firstcovalently bonded to the support surface as described above. Thereafter,the preformed polymer is dissolved in a suitable solvent in which it issoluble. However, the solid support surface remains in a solid form,i.e. it maintains the integrity of its shape, (e.g. at the inner wall ofthe capillary or particles). The relative solubility characteristics ofthe support surfaces and preformed polymers are well known.

Suitable free radical catalysts include ammonium persulfate, hydrogenperoxide, hydrazine and a20 based catalysts. Cross-linking of neutralpolymer coatings are formed by cross-linking polymers such as PVP,²³,24polystyrene or polyvinyl acetate²⁵ by treating with the above freeradical catalyst at elevated temperature. The general mechanism ofcross-linking with these free radical catalysts includes abstractinghydrogen atoms from the polymer chain by SO₄ ⁻ or OH⁻ radicals.²⁶

The molecular weight of the preformed polymer may be varied over a widerange so long as it is capable of being dissolved for uniform contactwith the support surface. Typical molecular weights may vary from 5,000to as high as 1,000,000 or more.

One of the features of the present invention is that under hydrogenabstraction conditions, the preformed polymer covalently links to thecoupling agent and simultaneously cross-links to form a coatingcomprising a three-dimensional polymer network on the support structure.This cross-linked nature provides high pH stability to the surfacecoating and inhibits nonspecific interactions of the analyte with thesurface. Cross-linking occurs in the coating and can be verified fromliterature on cross-linking of polymers. Experimental verification ofcross-linking was done by solution phase experiments where the linearpolymer dissolves in normally used solvents whereas the cross-linkedpolymers does not. High pH stability of this coating compared to linearpolymer coating is an indirect verification of cross-linking.

The hydrogen abstraction conditions according to the present inventionare the conditions well known for hydrogen abstraction from polymers.Encyclopedia of Polymer Science and Engineering, John Wiley & Sons, NewYork, (1990) Vol. 4, p.385;²⁴,25. The conditions include elevatedtemperatures for a sufficient holding time in the presence of a freeradical catalyst. By "elevated temperatures" means a temperature inexcess of room temperature, typically from a minimum of 40° C. to ashigh as 150° C. The temperature may vary with the labile characteristicsof the leaving group in the covalently bound coupling agent and in thepreformed polymer, the type of free radical catalyst, and the like. Thesolvent used will either enhance the formation of cross-linking bytransferring the low molecular weight radical to the polymer chain orthe solvent radical will minimize the formation of cross-links bycombining with macro radicals.

In a preferred embodiment fully formed polymers are coated onto thesurface of a fused silica capillary. No additional derivatization of thepolymer is required prior to coupling. The coating is achieved bypolymer macro-radicals formed during the cross-linking process. Thepolymer becomes attached to the surface of a silane treated capillarythrough the same radical mechanism. Cross-linking between the polymerchains and coupling to the silane takes place simultaneously. At aminimum, cross-linking occurs through bonding sites created on thepreformed polymer carbon backbones. However, additional cross-linkingmay take place through bonding sites pretreated on the preformed polymerside chains. The resulting coating is a highly cross-linked, stablelayer on the capillary surface. The pH stability of this coating isimproved over existing coatings, due to the high level of cross-linkingon the capillary surface. No specific leaving groups other than hydrogenare necessary on the silane or on the polymer for the coupling processto take place. However, the extent of substitution on the polymer chainand the silane, coupled with the solvent and the initiator system,²²determines the extent of cross-linking and coupling.

According to the invention, cross-linking of polymers, such as PVP,²³,24 polystyrene and polyvinylacetate²⁵ can be achieved by treating thepolymer with free radical initiators such as ammonium persulfate,hydrogen peroxide, and hydrazine. The general mechanism of cross-linkinginvolves abstracting hydrogen atoms from the polymer chain by SO₄ ⁻ orOH radicals.²⁶ The macroradicals formed then combine to formcross-links. Competing reactions include dissociation of the polymerchain, in some cases generating some few radical- and vinyl-terminatedpolymer chains. The solvent used will either enhance the formation ofcross-links by transferring the low molecular weight radical to thepolymer chain, or the solvent radical will minimize the formation ofcross-links by combining with macroradicals.

ORGANIC POLYMERIC SOLID SUPPORT EMBODIMENT

In the use of a suitable organic polymeric solid support, the coating ofthe present invention may be formed without a coupling agent. Thepolymeric solid support of the present invention has saturated orunsaturated carbon chains including leaving groups in the polymerbackbone or in side chains, including cross-links. The above discussionof "carbon chains", "saturated" or "unsaturated" and "leaving groups"for the preformed polymer apply to the organic polymeric solid support.In this instance, the polymeric support material has the requisitefunctional groups for covalently bonding directly to the preformedpolymer under hydrogen abstraction conditions without the requirementfor a separate coupling agent. In that regard, the carbon chains withthe functional groups may be present after polymerization of one or moremonomers to form a homopolymer or copolymer. For example, a suitablecopolymer would be the well known copolymer styrene-divinyl benzene usedas the resin particles in a packed bed for liquid chromatography. Inthis instance, the polymer includes the residual unsaturated linkagesfrom divinyl benzene.

Suitable monopolymers include homopolymers of polyvinylidene monomer.Suitable copolymers include copolymer of polyvinylidene monomer andmonovinylidene monomer.

In addition to copolymers, suitable polymeric support particles includepolymers with grafted side chains or block copolymers or any otherderivatized polymer so long as the polymeric support includes therequisite functional groups at the time of reaction with the preformedpolymer. Suitable substrates of this type include substrates containingsurface hydroxyl groups, for example, copolymers of divinylbenzene,styrene and vinylbenzylchloride, which are base esterified for reactingwith preformed polymer. In that regard, in the broad sense a couplingagent may be used to provide possible reactive sites on the organicpolymeric support prior to covalent bonding with the preformed polymer.Suitable bifunctional coupling agents which bind the organic polymericsupport particles to the preformed polymer include acrylic anhydrides,acryloyl chlorides, vinyl benzyl chlorides and the like. Reactants ofthis type are known to those skilled in the art and are listed ingeneral organic chemistry books such as Advanced Organic Chemistry,Reaction, Mechanism & Structure by Jerry March (John Wiley & Sons, Inc.1992).

The same hydrogen abstraction conditions discussed above regarding thecoupling agent bound inorganic solid supports generally apply to thisorganic polymer solid support. Also, the same types of preformedpolymers described above may be used as the preformed polymers in thisembodiment.

The polymer is attached through multiple linkages on the surface of thesubstrate, thus enhancing the pH stability of the coating. In the abovecoupling process, stringent control of reaction conditions is notrequired since the polymer is already formed. In situ polymerizedcoatings on the other hand, require stringent control of such variablesas oxygen levels and temperature²⁷ because the polymer has to be createdon the surface.

In order to illustrate the present invention, the following examples areprovided.

EXAMPLE 1 A 3-Methacryloxypropyltrimethoxysilane Coupled to PolyvinylPyrrolidone (PVP) Polymer

Capillary preparation: The inner wall of the capillary (formed ofsilica) is first rinsed with 1M NaOH for at least 10 minutes with anapplied pressure of 10 psi, followed by deionized water. The capillaryis then rinsed with 1% (w/v) acetic acid in water for 2 hours.

Silanization: A 1% solution (v/v) of3-methacryloxypropyltrimethoxysilane in 1% acetic acid is prepared andthe capillary is rinsed with this solution for 1 hour using 10 psipressure. The capillary is stored in the silane solution for at least 24hours and then displaced with deionized water.

Polymer solution preparation: A 4% PVP (MW: 360,000) solution in wateris prepared. 5 μl of TEMED and 50 μl of a 10% ammonium persulfate (w/w)is added to 10 ml of the above polymer solution. The above solution isthen pressurized into the silanized capillary by applying 10 psipressure.

Polymer bonding step: The capillary filled with the polymer solution isplaced in an oven with the ends sealed, and baked at 80° C. for 18hours. The capillary is rinsed with deionized water and is ready fortesting.

EXAMPLE 2 An Allyl Silane is Coupled to Polyvinyl Pyrrolidone (PVP)Preformed Polymer

All the steps are identical to Example 1 except the silane was anallyltrimethoxysilane.

EXAMPLE 3 Chlorodimethyloctyl Silane is Coupled to a PVP Polymer

All the steps are identical to Example 1 except the silane is preparedas a 2% solution in ethanol.

EXAMPLE 4 A 3-Methacryloxypropyltrimethoxysilane is Coupled toPolyacrylamide Preformed Polymer

All the steps are identical to Example 1 except the preferred polymer ispolyacrylamide and is prepared as a 2% solution in water.

EXAMPLE 5 A 3-Methacryloxypropyltrimethoxysilane Coupled to an AnionicPolyacrylamide Polymer

Conditions similar to example 4 except the preferred polymer is 2%anionic polyacrylamide.

EXAMPLE 6 A 3-Methacryloxypropyltrimethoxysilane is Coupled to aPreformed Cationic Polymer

All conditions are the same as Example 1 except the polymer is acopolymer of 2% (w/w) acrylamide and 1.8% (w/w)methacrylamidopropyltrimethylammonium chloride.

EXAMPLE 7 A 3-Methacryloxypropyltrimethoxysilane Coupled to PVP PolymerUsing a Different Free Radical Initiator

All the conditions are the same as example 1 except 4,4'-Azo-bis-(4-cyano pentanoic acid) was used instead of ammonium persulfateand TEMED. 61.5 μl of a 10% solution (w/w) of 4,4'- Azo-bis-(4-cyanopentanoic acid) in methanol was added to 10 ml of a 4% PVP polymersolution.

EXAMPLE 8 Anionic Polymer Attached to Silica Particles Suitable for Useas a Packing

A. Silica pretreatment 15 μm silica particles with 150 Å pore size(grade 215hp4X 1930) was obtained from Davisil. 30.15 g of deionizedwater was added to 4.77 g of the above silica material and disperseduniformly. 2.23 g of Conc. Nitric acid was added and the mixture washeated in an oven for 18 hours at 52° C. The material was removed afterthe heat treatment and washed with deionized water until the pH of thewater was measured to be neutral. After removal of water the materialredispersed in deionized water to a total weight of 54.14 g followed byaddition of 0.65 g of acetic acid and 0.5 g ofmethacryloxypropyltrimethoxy silane reagent. The material was disperseduniformly and heated in an oven for 18 hours at 52° C.

B. Preforming polymers: 0.98 g of 2-acrylamido-2-methyl propane sulfonicacid (AMPS) (Monomer Polymer & DAJAC Laboratories, Pa., USA) and 0.98 gof acrylamide (BDH Laboratories, Poole, England) was dissolved in 16.6 gof deionized water and degassed. 0.024 g of VA 086 Azo initiator (WakoPure Chemical Industries Ltd., Japan) was added to the above monomermixture and the mixture was placed in an oven at 52° C. for 18 hours.The resulting polymer was a copolymer of AMPS and acrylamide. The abovepolymer was weighed, followed by precipitation with suitable solvent.The precipitated polymer was then redissolved and reconstituted to theoriginal weight in deionized water.

C. Attachment to silica particle: 18 g of the pretreated silica slurryfrom step A was washed with deionized water, acetone and followed bydeionized water. The final weight of the silica material was adjusted to10 g with deionized water 1 g of the polymer from step B was added tothe above and 0.02 g of VA 086 Azo initiator (Wako Pure ChemicalIndustries Ltd., Japan) was added. The entire material was dispersedhomogeneously and then placed in an oven at 80° C. for 18 hours. Theresulting covalently bonded material from above was washed with waterfollowed by 100 mM acetic acid before packing in an analytical columnusing standard methods and apparatus at 6000 psi for 10 minutes. Thissilica bonded polymer column is suitable for chromatographic separationsof inorganic cations and other macromolecules. The above silica bondedpolymeric material when packed in capillaries is suitable forelectrochromatographic separations of inorganic cations and othermacromolecules.

EXAMPLE 9 Anionic Polymer is Attached to Polymeric Particles Suitablefor Use as a Packing

A. 2.4 g of a dried 55% cross-linked macroporous resin (substrate isethylvinylbenzene cross-linked with 55% divinylbenzene, resinpreparation described in U.S. Pat. No. 4,224,415) was dispersed in 4 gof tetrahydrofuran and 5 g of water was added to this slurry. 1 g ofpolymer solution prepared as shown in Example 9 step B was added to thisslurry. 0.02 g of VA 086 Azo initiator (Wako Pure Chemical IndustriesLtd., Japan) was added and the entire material was dispersedhomogeneously and then placed in an oven at 80° C. for 18 hours. Theresultant polymeric material from above was washed with water followedby 100 mM acetic acid before packing in an analytical column usingstandard methods and apparatus at 6000 psi for 10 minutes. Thispolymeric column is suitable for chromatographic separations ofinorganic cations and other macromolecules. The above polymeric materialwhen packed in capillaries is suitable for electrochromatographicseparations of inorganic cations and other macromolecules.

EXAMPLE 10 Cationic Polymer Attached to Silica Particles Suitable forUse as a Packing

A. Preforming polymers: 0.98 g of methacrylamidopropyltrimethyl-ammoniumchloride and 0.98 g of acrylamide (BDH Laboratories, Poole, England) wasdissolved in 16.6 g of deionized water and degassed. 0.024 g of VA 086Azo initiator (Wako Pure Chemical Industries Ltd., Japan) was added tothe above monomer mixture and the mixture was placed in an oven at 52°C. for 18 hours. The resulting polymer was a copolymer ofmethacrylamidopropyltrimethyl-ammonium chloride and acrylamide. Theabove polymer was weighed, followed by precipitation with suitablesolvent. The precipitated polymer was then redissolved and reconstitutedto the original weight in deionized water.

Attachment to silica particle: 18 g of pretreated silica from step A inExample 8 was washed with deionized water, acetone and followed bydeionized water. The final weight of the silica material was adjusted to10 g with deionized water. 1 g of polymer solution from step A inExample 10 was added to the silica material in water followed by 0.02 gof VA 086 Azo initiator (Wako Pure Chemical Industries Ltd., Japan) Theentire material was dispersed homogeneously and then placed in an ovenat 80° C. for 18 hours. The resulting polymer bonded material from abovewas washed with water followed by 100 mM citrate buffer at pH 6.0 beforepacking in an analytical column using standard methods ant apparatus at6000 psi for 10 minutes. This silica bonded polymer column is suitablefor chromatographic separation of inorganic anions and othermacromolecules. The above polymer bonded silica material when packed incapillaries is also suitable for electrochromatographic separation ofinorganic anions and other macromolecules.

EXAMPLE 11 Cationic Polymer is Attached to Polymeric Particles Suitablefor Use as a Packing

A. 2.4 g of a dried 55% cross-linked macroporous resin (substrate isethylvinylbenzene cross-linked with 55% divinylbenzene, resinpreparation described in U.S. Pat. No. 4,224,415) was dispersed in 4 gof tetrahydrofuran and 5 g of water was added to this slurry. 1 g ofpolymer prepared as shown in Example 10 step A was added to this slurry.0.02 g of VA 086 Azo initiator (Wako Pure Chemical Industries Ltd.,Japan) was added and the entire material was dispersed homogeneously andthen placed in an oven at 80° C. for 18 hours. The resultant polymericmaterial from above was washed with water and 100 mM sodium carbonate atpH 11 before packing in an analytical column using standard methods andapparatus at 6000 psi for 10 minutes. This column is a polymeric columnsuitable for chromatographic separation of inorganic anions and othermacromolecules. The above polymeric material when packed in capillariesis also suitable for electrochromatographic separation of inorganicanions and other macromolecules.

EXAMPLE 12 Nonionic Polymer is Attached to Silica Particles Suitable forUse as a Packing

A. A 10% solution of polyvinylalcohol 25K (98% hydrolyzed fromPolyscience Laboratories) was prepared in water.

B. 18 g of pretreated silica as shown in step A in Example 9 wasprepared and washed with deionized water, acetone and followed bydeionized water. The final weight of the silica material was adjusted to10 g with deionized water. 1 g of polymer solution from step A was addedto the silica material in water followed by 0.02 g of VA 086 Azoinitiator (Wako Pure Chemical Industries Ltd., Japan). The entirematerial was dispersed homogeneously and then placed in an oven at 80°C. for 18 hours. The resulting polymer bonded material from above waswashed with water followed by 100 mM acetic acid before packing inanalytical column using standard methods and apparatus at 6000 psi for10 minutes. This column is suitable for normal phase chromatographicseparations and in size exclusion applications. The above polymer bondedsilica material when packed in capillaries is suitable forelectrochromatographic separations.

EXAMPLE 13 Nonionic Polymer Attached to Polymeric Particles Suitable forUse as a Packing

A. 2.4 g of a dried 55% cross-linked macroporous resin (substrate isethylvinylbenzene cross-linked with 55% divinylbenzene, resinpreparation (resin preparation described in U.S. Pat. No. 4,224,415) wasdispersed in 4 g of tetrahydrofuran and 5 g of water was added to thisslurry. 1 g of polymer prepared as shown in Example 12 step A was addedto this slurry. 0.02 g of VA 086 Azo initiator (Wako Pure ChemicalIndustries Ltd, Japan) was added and the entire material was dispersedhomogeneously and then placed in an oven at 80° C. for 18 hours. Theresultant polymeric material from above was washed with water and 100 mMacetic acid before packing in an analytical column using standardmethods and apparatus at 6000 psi for 10 minutes. This column is apolymeric column suitable for size exclusion separations. The abovepolymeric material is suitable for electrochromatographic separations.

EXAMPLE 14 Cationic Polymer is Attached to Polymer Particles Suitablefor Use as a Packing

A. Preforming polymers: To 4.16 g of2-methacryloxyethyltrimethylammonium chloride (70% in H20 fromPolysciences Laboratories) was added 3.92 g of methanol and degassedprior to adding 0.03 g of VA 044 Azo initiator (Wako Pure ChemicalIndustries Ltd., Japan). The above mixture was placed in an oven for 18hours at 52° C. to form a polymer of the above monomer.

B. Attachment to a polymeric particle: 0.4 g of polymer prepared asshown in step A was added to 5.1 g of water. The above mixture wasinitiated by adding 0.02 g of Azobiscyanovaleric acid initiator (FlukaChemicals). 2.3 g of acetic acid is added to above mixture followed by2.31 g of a dried 55% cross-linked macroporous resin (substrate isethylvinylbenzene cross-linked with 55% divinylbenzene resin preparationdescribed in U.S. Pat. No. 4,224,415) and dispersed. 4.7 g of ammoniumhydroxide was added to above slurry and dispersed homogeneously and thenplaced in an oven at 52° C. for 8 hours. The resultant polymericmaterial from above was washed with 50 ml of deionized water, followedby 200 ml of acetone and followed by 50 ml of a 1×carbonate/bicarbonatesolution (1.8 mM of sodium carbonate +1.7 mM of sodium hydrogencarbonate) and dispersed in a 10×carbonate/bicarbonate solution (18 mMsodium carbonate/17 mM sodium hydrogen carbonate) before packing in ananalytical column using standard methods and apparatus at 6000 psi for10 minutes. This column is a polymeric column suitable forchromatographic separation of inorganic anions and other macromolecules.The above polymeric material when packed in capillaries is also suitablefor electrochromatographic separation of inorganic anions and othermacromolecules.

EXAMPLE 15 Cationic Copolymer is Attached to Polymeric ParticlesSuitable for a Use as a Packing

A. Preforming polymers: To 2.12 g of2-methacryloxyethyltrimethylammonium chloride (70% in H20 fromPolysciences Laboratories) was added 1.7 g of Vinylbenzylchloride (DowChemicals) and 5.67 g of methanol was added to this mixture and thesolution was mixed and degassed prior to adding 0.02 g of VA 044 Azoinitiator (Wako Pure Chemical Industries Ltd., Japan). The above mixturewas placed in an oven for 18 hours at 52° C. to form a copolymer of theabove monomers.

B. Attachment to a polymeric particle: 0.45 g of polymer prepared asshown in step A was added to 5.3 g of water. The above mixture wasinitiated by adding 0.02 g of Azobiscyanovaleric acid initiator (FlukaChemicals). 2.37 g of acetic acid is added to above mixture followed by23 g of a dried 55% cross-linked macroporous resin (resin preparationdescribed in U.S. Pat. No. 4,224,415) and dispersed. 4.9 g of ammoniumhydroxide was added to above slurry and dispersed homogeneously and thenplaced in an oven at 52° C. for 8 hours. The resultant polymericmaterial from above was washed with 50 ml of deionized water, followedby 200 ml of acetone and followed by 50 ml of a 1×carbonate/bicarbonatesolution (1.8 mM of sodium carbonate +1.7 mM of sodium hydrogencarbonate) and dispersed in a 10×carbonate/bicarbonate solution (18 mMsodium carbonate/ 17 mM sodium hydrogen carbonate) before packing in ananalytical column using standard methods and apparatus at 6000 psi for10 minutes. This column is a polymeric column suitable forchromatographic separation of inorganic anions and other macromolecules.The above polymeric material when packed in capillaries is also suitablefor electrochromatographic separation of inorganic anions and othermacromolecules.

EXAMPLE 16

The separation of basic proteins in several polymer coated capillariesusing the above bonding approach and a 50 mM sodium acetate buffer (pH4.5) is shown in FIG. 1. FIG. 1 shows separations of basic proteinsusing various polymer coated capillaries. Capillary: 50 cm total length;45 cm to detector; 50 μm i.d. Buffer: 50 mM sodium acetate at pH 4.5.Conditions: 20 kV (400 V/cm); Gravity Injection: 50 mm×10 s. Detection:V, 210 nm. Sample concentration: 100 μg/ml. Peak identification: (1)Lysozyme (Chicken Egg White), (2) Cytochrome c (Bovine Heart), (3)Ribonuclease A (Bovine Pancreas), (4) Myoglobin (Horse Skeletal Muscle),(5) a-Chymotrypsinogen A (Bovine Pancreas).

The basic proteins show approximately the same migration times in allthe capillaries. The capillary-to-capillary migration time variation was3.12% (RSD), indicating a small EO flow variation between thecapillaries. The efficiency generated in capillaries treated with (a) 4%PVP on MET silane, (b) 4% PVP on octyl silane, and (c) 2% polyacrylamideon MET silane were similar (app. 500,000 plates/50 cm). We observed noband broadening effects or efficiency loss due to hydrogen bondinginteractions with the polymer for the above polymer coatings, contraryto the observations of Zhao et al.⁶ and Strege and Lagu⁹. Much lowerefficiencies were generated in a PVP-coated-on-allyl silane capillary(160,000 plates/50 cm) and PEO on a MET silane (300,000 plates/50 cm).Hydrophobicity of the silane is probably not contributing to the loss ofefficiency in the PVP-allyl silane coated capillary, because higherefficiencies were realized with an even more hydrophobic octyl silane onthe PVP-octyl silane coated capillary. Although all the above coatedcapillaries produced a low EO flow, not all the silanols were modifiedby the coating process. Hence, performance of the coating was probablyrelated to the extent of shielding of analytes from residual silanols.The extent of modification of the surface silanols by the primary silanelayer, coupled with the level of coverage by the polymeric layerattached to the primary silane layer, determines the extent ofshielding. These results suggest that both the silane and the polymerinfluenced the coating performance.

EXAMPLE 17

The stability of a MET-PVP coating was tested by running 500 repetitiveanalyses of a basic protein test mixture. The RSD of migration times waswithin 2% (n=500). The corrected migration times with respect tolysozyme (plotted in FIG. 2) showed an RSD of 0.42% (n=500). FIG. 2 is areproducibility study using a MET-PVP (360K) coated capillary. All otherconditions are the same as in FIG. 1.

EXAMPLE 18

The coating was further tested for interaction with basic proteins undera variety of concentration conditions. The response versus concentrationplots showed good linearity, and correlation coefficients (r²) higherthan 0.995 were obtained for all the tested proteins. The capillarygenerated more than 250,000 plates at a concentration of 300 μg/ml andmore than 100,000 plates at a concentration of 1 mg/ml. The RSD inmigration times in the tested range of 5 μg/ml to 1000 μg/ml was 1.6%,consistent with the long term performance of this capillary. Theconcentration detection limit for basic proteins under our experimentalconditions was 5 μg/ml. Based on the linearity of response versusconcentration curves, high efficiency under high concentrationconditions, low detection limit, and minimal migration time variationunder high analyte concentrations, we conclude that the analytesinteracted minimally with the coating surface.

The MET-PVP capillary remained stable under a variety of buffer and pHconditions. The EO mobility in this coated capillary was less than2×10⁻⁵ cm² /V.s at pH 10 using a 10 mM borate buffer and tested for morethan 48 hours. A fused silica capillary tested under the same conditionsgave an EO mobility of 62.5×10⁻⁵ cm² /V.s. The EO flow was substantiallyreduced by the polymer coating. The EO mobility in the polymer coatedcapillary increased from 1.5×10⁻⁵ cm² /V.s to 5×10-5 cm² /V.s after 40 husing a 50 mM sodium carbonate buffer at pH 11. At extremes of pH, thesilanols slowly become exposed due to some coating loss. This is not anunusual finding, given the lack of stability of the Si--O--Si linkageand the dissolution of silica at high pH. In performing the pH stabilitystudies, we observed that electrophoretic runs were important toestablish stability rather than mere contact with the run buffer. Theoctylsilane-PVP capillary also remained stable at pH 10, and gave an EOmobility of <2.5×10⁻⁵ cm² /V.s. The high stability of our coating is dueto the high level of cross-linking on the surface of the capillary.

EXAMPLE 19

The effect of polymer size and concentration on the coating performancewas studied by coating several capillaries under the same conditions andtesting them for basic protein separations using a 50 mM sodium acetatebuffer at pH 4.5. (See Table 1 and FIG. 3). FIG. 3 shows the effect ofpolymer molecular weight and concentration on efficiency of basicproteins. All other conditions are the same as in FIG. 1.

                  TABLE I                                                         ______________________________________                                        Effect of Polymer Size and Concentration on Migration Time                    Average Migration Time in Minutes (n = 3)                                     Polymer size             Ribo-                                                and      Lyso-  Cyto-    nuclease                                                                             Myo-  α-Chymotryp-                      Concentration                                                                          zyme   chrome c A      globin                                                                              sinogen                                 ______________________________________                                        PVP 10K 4%                                                                             9.11   9.49     12.37  12.92 15.08                                   PVP 360K 9.35   9.81     12.87  13.3  15.60                                   4%                                                                            PVP 1M 4%                                                                              9.39   9.86     12.81  13.34 15.65                                   PVP 360K 9.15   9.6      12.45  13    15.20                                   1%                                                                            PVP 360K 9.33   9.78     12.72  13.26 15.56                                   10%                                                                           % RSD    1.37   1.61      1.76   1.45  1.68                                   ______________________________________                                    

The average efficiency was almost the same based on an RSD of less than5%. The RSD in migration times for the various proteins was less than2%, indicating that polymer size and concentration had a minimalinfluence on the coating performance. These results also indicated thatthe EO flow and shielding of surface silanols were nearly identical inthese capillaries. Testing under alkaline conditions using a phosphatebuffer at pH 8 gave an average EO flow of <1.7×10⁻⁵ cm² /V.s for all thecapillaries (tested for more than 24 hours). The coating process wasinsensitive to the molecular weight change and concentration of thepolymer. No optimization of the polymer size or concentration wasrequired to achieve optimal performance, unlike the approach of Zhao etal.¹⁶ Additionally, the coating thickness was optimal based on the highefficiencies observed using these coatings.

EXAMPLE 20

Replacing the persulfate initiator system with an azo-based initiatorsystem resulted in minimal change in performance for the MET-PVPcapillary. The average efficiency for basic proteins in a MET-PVP coatedcapillary using an azo initiator was ˜500,000 plates/capillary. Themigration time of the basic proteins using a persulfate initiator systemwas nearly identical to the azo-based initiator system based on an RSDof 1.1% (n=6), confirming a free radical-based coupling mechanism of thepolymer to the silane.

EXAMPLE 21

The reproducibility of the coating process was studied by coating fivecapillaries in parallel with MET-PVP coating. The capillaries gave anRSD of less than 2.5% in migration times for the basic proteins andconfirmed the reproducibility of the coating protocol.

EXAMPLE 22

Cationic coatings. In addition to attaching neutral polymers, the abovecoupling process was found suitable for attaching cationic polymers tothe capillary surface as shown in Example 6. The cationic polymer coatedcapillary had an EO flow directed toward the anode. Separation of acidicproteins with average efficiencies of >200,000 plates/50 cm wasaccomplished using a cationic polymer coated capillary and 25 mMphosphate buffer at pH 7 (FIG. 4). FIG. 4 shows acidic proteinseparations using a cationic polymer coated capillary. Capillary: 50 cmtotal length: 45 cm to detector, 50 μm i.d. Buffer: 25 mM sodiumphosphate at pH 7. Conditions: 20 kV (400 V/cm); Gravity Injection: 50mm×10 s. Detection: UV, 210 nm. Sample concentration: 100 μg/ml. Peakidentification (1) α-Lactalbumin (Bovine Milk), (2) Carbonic anhydrase(Bovine Erythrocytes), (3) Myoglobin (Horse Skeletal Muscle). Fastseparations of acidic proteins were possible since the anionic proteinsmigrated with the EO flow.

EXAMPLE 23

The capillary was also useful in separating a standard mixture of seveninorganic anions (FIG. 5). FIG. 5 shows separation of test anions usinga cationic polymer coated capillary. Capillary: 50 cm total length; 45cm to detector, 50 μm i.d. Buffer: 1.6 mM Triethanolamine, 2.25 mMpyromellitic acid adjusted to pH 7.7 with 1N NaOH. Conditions: -20 kV(400 V/cm); Gravity Injection: 100 mm ×30 s. Detection: Indirect V, 250nm. Sample concentration: 1 μg/ml. Peak identification: (1) Bromide, (2)Chloride, (3) Sulfate, (4) Nitrite, (5) Nitrate, (6) Fluoride, (7)Phosphate.

The seven ions were baseline resolved using a pyromellitic acidcontaining buffer with efficiencies ranging from 30,000 plates/50 cm forfluoride to 140,000 plates/50 cm for nitrate. Analysis of the same ionsusing fused silica capillaries and indirect UV detection requiredspecial additives coupled with special pretreatment steps to achieveflow reversal in the anodic direction.

EXAMPLE 24

Coupling mechanism. The following solution phase and CE experiments wereperformed to understand the mechanism of coupling the polymer to thesilane.

CE Experiments. Several capillaries were coated under various conditions(Table II) using a 4% PVP polymer (MW 360K). Only the silane treatedcapillaries coated in the presence of the polymer, initiators and athermal treatment were stable, suggesting a covalent attachment of thepolymer to the MET silane through a hydrogen abstraction mechanism.

                  TABLE II                                                        ______________________________________                                        Effect of Capillary Coating Format on Stability                               Polymer Coating          Stability at pH 8                                    ______________________________________                                        Fused silica treated with 4% PVP 360K polymer and                                                      Not stable                                           initiators followed by thermal treatment for 18 h                             at 80° C.                                                              Silanized capillary treated with 4% PVP 360K poly-                                                     Not stable                                           mer at room temperature                                                       Silanized capillary treated with 4% PVP 360K poly-                                                     Not stable                                           mer and thermal treatment for 18 h at 80° C.                           Silanized capillary treated with 4% PVP 360K poly-                                                     Not stable                                           mer, initiators at room temperature                                           Silanized capillary treated with 4% PVP 360K poly-                                                     Stable and                                           mer, initiators and thermal treatment for 18 h                                                         Generated                                            at 80° C.         Reproducible                                                                  EO flow                                              ______________________________________                                    

Literature on grafting vinyl²⁸ silanes and vinyl monomers²⁹ into polymerchains supports the covalent linkage of the polymer to the silanethrough double bonds on the silane. Similarly, linkage of the polymer tothe silane through free radical sites created by hydrogen abstraction onother sites such as a α-carbon atom adjacent to the double bond is alsopossible.³⁰,31 Solution phase experiments were conducted to gain betterunderstanding of the coupling mechanism.

EXAMPLE 25

Solution phase experiments. Various polymer solutions were prepared inthe presence and absence of the added silane, and monitored after 18 h.The results are shown in Table III.

                  TABLE III                                                       ______________________________________                                        Solution Phase Experiments                                                    Mixture       Physical Appearance                                                                          Viscosity                                        ______________________________________                                        PVP 2% (360K) in water                                                                      Clear          Low, 6 centipoise                                PVP 2% (360K) in water +                                                                    Clear          Low, 6 centipoise                                initiators @ RT                                                               PVP 2% (360K) in water +                                                                    Clear but with slight                                                                        Low, 7 centipoise                                initiators @ 80° C.                                                                  yellowish coloration                                            PVP 2% (360K) in water +                                                                    Turbid milky material                                                                        High (>1000's of                                 initiators + 1% methacryl-                                                                  showing gelation; bottom                                                                     centipoise)                                      oxypropyl trimethoxy                                                                        of flask shows increased                                        silane @ 80%  gel attachment                                                  PVP 2% (360K) in water +                                                                    Milky material; turbidity                                                                    Higher than                                      initiators + 5% methacryl-                                                                  increases with excessive                                                                     previous sample                                  oxypropyl trimethoxy                                                                        gelation; flask bottom                                          silane @ 80%  shows excessive attach-                                                       ment of gel; precipitated                                                     material also interspersed                                                    in gel                                                          1% methacryloxypropyl                                                                       Turbid/milky water-like                                                                      Similar to water                                 trimethoxy silane +                                                                         material with precipitated                                      initiators @ 80° C.                                                                  particulate material                                                          attached to bottom                                                            of flask                                                        PVP 2% (360K) in acetic                                                                     Turbid low viscosity                                                                         10 centipose                                     acid + initiators + 1%                                                                      material; appears as latex                                                                   viscosity                                        methacryloxypropyl tri-                                                                     phase; some spotting in                                         methoxy silane @ 80° C.                                                              bottom of flask; no                                                           gelation                                                        ______________________________________                                    

PVP in the presence of silane and initiators when heated at 80 ° C.showed gelation and high viscosity, indicating cross-linking of thepolymeric material. Several reactions occurred simultaneously: (1)homopolymerization of the silane through the vinyl groups, (2) thesilane cross-linking with itself through condensation, (3) the silanecross-linking with the polymer, and (4) the polymer cross-linking withitself. Reaction (1) was minimal, as evidenced by no increase inviscosity when the silane was reacted with initiators in the absence ofthe polymer. Similarly, reaction (4) did not contribute to the hugeincrease in viscosity, since the polymer by itself showed a minimalincrease in viscosity. Contribution of silane condensation to theviscosity of the matrix was expected to be minimal, based on the minimalincrease in viscosity when the reaction was performed in acetic acid.

EXAMPLE 26

This was further tested by treating the materials obtained from theabove experiment with sodium fluoride and NaOH. (See Table IV forresults.)

                  TABLE IV                                                        ______________________________________                                        Effect of Alkali and Fluoride Treatment                                       Mixture          Appearance                                                   ______________________________________                                        PVP 2% (360K) in water +                                                                       Clear; retained clarity after treatment                      initiators @ 80° C.                                                                     with 1N NaOH                                                 PVP 2% (360K) in water +                                                                       Milky material; retained gel-like                            initiators + 1% methacryloxy-                                                                  appearance after treatment with                              propyl trimethoxy silane @ 80° C.                                                       1N NaOH (solution monitored at pH                                             12 and 13.2 for 1 week); gel-like                                             appearance retained when 10% NaF                                              was added and heated @ 50° C.                                          for 18 h                                                     PVP 2% (360K) in acetic acid +                                                                 Milky material formed clear solution                         initiators + 1% methacryloxy-                                                                  on treatment with 1N NaOH                                    propyl trimethoxy silane @ 80° C.                                      methacryloxypropyl trimethoxy                                                                  Turbid milky material; dissolved and                         silane + .initiators @ 80° C.                                                           formed clear solution when 1N NaOH                                            was added.                                                   ______________________________________                                    

Treatment with fluoride ion is expected to inhibit silanecondensation.³² Similarly, extreme alkaline environment (above pH 10)inhibits silane condensation.³³ Treatment of the gel-like material(mixture of PVP, silane, initiator treated at 80° C.) with sodiumfluoride and heating the mixture at 50 ° C. for 18 h showed no change inthe solution. The retention of gel-like behavior by the PVP-silanemixture suggests a covalent cross-linking reaction between the polymerand the silane. Similarly, treating with 1N NaOH also showed no changein the PVP-silane mixture, further reaffirming the above result. Thecontribution from silane condensation to the huge increase in viscositywas minimal based on the results from NaOH treatment of the silanemixture and the PVP-silane mixture (in acetic acid). These resultsconfirm that the silane cross-links with the polymer through a hydrogenabstraction mechanism.

EXAMPLE 27

We performed solution phase experiments, simulating coupling of PVP toan octylsilane by thermal treatment of PVP in the presence of anin-house synthesized trimethyldecylsilane reagent and initiators indichloromethane solvent. A highly cross-linked gel-like material wasformed at the bottom of the flask after the 18 hour thermal treatmentstep. PVP polymer with added initiators and PVP in the presence oftrimethyldecylsilane with no added initiators did not form a gel-likematerial under the same conditions. Trimethyldecylsilane has no reactivefunctional groups for chemical coupling reactions. The above experimentsuggests the formation of a covalent cross-link between PVP andtimethyldecylsilane through a hydrogen abstraction mechanism.

EXAMPLE 28

Analysis of Milk Proteins. Separation of milk proteins (acidic proteins)became possible due to the low EO flow generated in the MET-PVPcapillary. Published CE methods for analyzing milk proteins involveworking at low buffer pH conditions with added polymer additives.³⁴ Dueto the high pH stability of this coating, we attempted the aboveseparations without adding any polymer additives at pH 8.4. Vitamin Dmilk was centrifuged at 8000 RPM for 4 minutes and incubated in areduction buffer following a sample preparation procedure by Jong etal.³⁴ Excellent separation of the whey proteins from casein wasachieved, as shown in FIG. 6. FIG. 6 shows separation of proteins from2% Vitamin D Milk using a MET-PVP coated capillary. Capillary: 50 cmtotal length; 45 cm to detector; 50 μm i.d. Buffer: 100 mM sodiumphosphate, pH 8.4 with 6M urea. Conditions: -30 kV (600 V/cm); GravityInjection: 150 mm×30 s. Detection: UV, 210 nm. Peak identification: (1)α-Lactalbumin, (2) β-Lactoglobulin A & B, (3) α-Caseins (4) κ-Caseins.(5/6) β-Caseins.

Individual proteins were identified by running standard samples. Theseseparations agreed well with the findings of Jong et al.³⁴ More than 50runs were run in this capillary using various milk samples, and thecapillary performed reliably, with no loss of separation efficiency.

EXAMPLE 29

Analysis of Hemoglobin variants. Baseline resolution of the four commonvariants of hemoglobin was achieved using a PVP-MET coated capillarywith >650,000 plates/65-cm. The two commonly occurring normalhemoglobins are adult hemoglobin (HbA) and fetal hemoglobin (HbF).Sickle cell hemoglobin (HbS) is one of the abnormal hemoglobins, inwhich a single replacement of glutamic acid with valine occurs inposition 6 of the beta chain, thus altering the solubility of thisprotein. In β-thalessemia HbC, the individual has lysine instead ofglutamic acid in position 6 on their beta chain. More than 50 runs ofthese samples were run without any loss in separation efficiency orperformance. The unidentified leading component was present in all thehuman Hb samples. These separations were far superior to those shown inthe literature.³⁵

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What is claimed is:
 1. A method of coating a solid support surface whichalters the properties of the support surface for separating solubleanalytes in a fluid stream, said method comprising(a) covalently bindinga coupling agent including functional groups, capable of forming freeradicals under hydrogen extraction conditions in a layer on said supportsurface, and (b) thereafter, contacting said covalently bound couplingagent with a solution of said preformed polymer comprising totallysaturated, substituted or unsubstituted carbon chain backbones includingleaving groups under hydrogen abstraction conditions of elevatedtemperature in the presence of a free radical catalyst, said supportsurface being substantially insoluble in said solvent, to remove leavinggroups from said preformed polymer carbon chain backbones to form freeradical carbon binding sites and to convert at least some of saidfunctional groups to free radicals thereby covalently bonding said freeradical carbon binding sites to said coupling agent functional groupfree radicals and to crosslink at least some of said preformed polymerthrough thus-formed free radical carbon binding sites therein to form athree-dimensional polymer network coating on said solid support surface.2. The method of claim 1 in which said preformed polymer leaving groupscomprise halogens.
 3. The method of claim 1 in which said preformedpolymer leaving groups comprise hydrogen.
 4. The method of claim 1 inwhich said coupling agent comprises at least one carbon chain with atleast one leaving group.
 5. The method of claim 4 in which at least partof said covalent bonding of said coupling agent to said preformedpolymer is by leaving group abstraction from said coupling agent carbonchain.
 6. The method of claim 1 in which said coupling agent layercomprises at least one carbon chain backbone with at least oneunsaturated carbon to carbon linkage and at least a portion of saidcovalent bonding is by a free radical addition reaction between said onecarbon to carbon linkage and said preformed polymer.
 7. The method ofclaim 1 in which said support surface comprises silica and said couplingagent binds to said support surface through Si--O--Si linkages.
 8. Themethod of claim 1 in which said preformed polymer is selected from thegroup consisting of PVP, polyacrylamide, polyethylene oxide andpolyvinylalcohol.
 9. The method of claim 1 in which said support surfacecomprises silica and said coupling agent comprises a silane.
 10. Themethod of claim 1 in which said coupling agent is bound to said solidsupport surface by linkages selected from the group consisting of Si--C,Si--O--C, or Si--N.
 11. The method of claim 1 in which at least some ofsaid coupling agent is unbound in step (a) and said unbound couplingagent is removed from said solid support surface prior to step (b). 12.The method of claim 1 in which said coupling agent comprises a carbonchain and said leaving group comprises hydrogen which is abstracted fromsaid carbon chain, and the covalent bonding of said coupling agent andpreformed polymer is between hydrogen abstracted sites on said couplingagent and said preformed polymer.
 13. The method of claim 1 in whichsaid solid support surface comprises the inner wall of a fluid conduit.14. The method of claim 13 in which said fluid conduit comprises acapillary suitable for capillary electrophoresis.
 15. The method ofclaim 1 in which said solid support comprises the packing of aflowthrough particle bed.
 16. The method of claim 1 in which said solidsupport surface is formed of a material selected from the groupconsisting of silica, quartz, glass, alumina, titanic, thoria, zirconia,and beryllia.
 17. A method of coating a polymeric solid support surfacewith a surface having saturated or unsaturated carbon chains includingleaving groups, said coating altering the properties of the supportsurface for separating components in a fluid stream, said methodcomprising contacting said support surface with preformed polymercomprising totally saturated carbon chain backbones under hydrogenabstraction conditions of elevated temperature in the presence of a freeradical catalyst, to abstract hydrogen from said preformed polymercarbon chain backbones to form hydrogen abstracted carbon sites whichcovalently bond said support surface carbon chain backbones, and tocrosslink at least some of said preformed polymer through hydrogenextraction carbon sites therein to form a three-dimensional polymernetwork coating on said solid support surface.
 18. The method of claim17 in which at least part of said covalent bonding is through saidsupport surface carbon chains by hydrogen extraction therefrom.
 19. Themethod of claim 17 in which said preformed polymer is formed byprepolymerizing a monomer including an unsaturated carbon-to-carbonlinkage.
 20. The method of claim 17 in which said preformed polymer isselected from the group consisting of PVP, polyacrylamide, polyethyleneoxide and polyvinylalcohol.
 21. The method of claim 17 in which theseparation is by capillary electrophoresis and said conduit is acapillary.
 22. The method of claim 17 in which hydrogen is abstractedfrom said support surface carbon chains and the bonding thereof withsaid preformed polymer is through hydrogen abstraction sites.
 23. Themethod of claim 17 in which said solid support surface comprises theinner wall of a fluid conduit.
 24. The method of claim 17 in which saidfluid conduit comprises a capillary suitable for capillaryelectrophoresis.
 25. The method of claim 17 in which said solid supportcomprises the packing of a flowthrough particle bed.